Targeting KRAS Regulation with PolyPurine Reverse Hoogsteen Oligonucleotides
KRAS is a GTPase involved in the proliferation signaling of several growth factors. The KRAS gene is GC-rich, containing regions with known and putative G-quadruplex (G4) forming regions. Within the middle of the G-rich proximal promoter, stabilization of the physiologically active G4<sub>mid&...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2022-02-01
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| Series: | International Journal of Molecular Sciences |
| Subjects: | |
| Online Access: | https://www.mdpi.com/1422-0067/23/4/2097 |
| Summary: | KRAS is a GTPase involved in the proliferation signaling of several growth factors. The KRAS gene is GC-rich, containing regions with known and putative G-quadruplex (G4) forming regions. Within the middle of the G-rich proximal promoter, stabilization of the physiologically active G4<sub>mid</sub> structure downregulates transcription of KRAS; the function and formation of other G4s within the gene are unknown. Herein we identify three putative G4-forming sequences (G4FS) within the <i>KRAS</i> gene, explore their G4 formation, and develop oligonucleotides targeting these three regions and the G4<sub>mid</sub> forming sequence. We tested Polypurine Reverse Hoogsteen hairpins (PPRHs) for their effects on <i>KRAS</i> regulation via enhancing G4 formation or displacing G-rich DNA strands, downregulating <i>KRAS</i> transcription and mediating an anti-proliferative effect. Five PPRH were designed, two against the <i>KRAS</i> promoter G4<sub>mid</sub> and three others against putative G4FS in the distal promoter, intron 1 and exon 5. PPRH binding was confirmed by gel electrophoresis. The effect on <i>KRAS</i> transcription was examined by luciferase, FRET Melt<sup>2</sup>, qRT-PCR. Cytotoxicity was evaluated in pancreatic and ovarian cancer cells. PPRHs decreased activity of a luciferase construct driven by the <i>KRAS</i> promoter. PPRH selectively suppressed proliferation in KRAS dependent cancer cells. PPRH demonstrated synergistic activity with a <i>KRAS</i> promoter selective G4-stabilizing compound, NSC 317605, in KRAS-dependent pancreatic cells. PPRHs selectively stabilize G4 formation within the KRAS mid promoter region and represent an innovative approach to both G4-stabilization and to <i>KRAS</i> modulation with potential for development into novel therapeutics. |
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| ISSN: | 1661-6596 1422-0067 |